Endothelial colony forming cells (ECFCs) are rare circulating endothelial cells, particularly abundant in umbilical cord blood, with clonal proliferative potential and intrinsic in vivo vessel forming ability1-6. ECFCs, also called blood outgrowth endothelial cells (BOEC)7, have been shown to be directly transplantable in sex-mismatched human bone marrow transplant patients, with the most proliferative circulating BOEC displaying genetic markings of the donor marrow7, 8. It is not understood what type of cell within donor marrow gives rise to ECFCs. When cultured ECFCs are injected intravenously into pre-clinical rodent vascular injury models, they are rapidly recruited the site of vascular injury or tissue ischemia to orchestrate initiation of a vasculogenic response9-11. Human ECFCs have been reported to enhance vascular repair and improve blood flow following myocardial infarction12, 13, stroke9, ischemic retinopathy14, 15, ischemic limb injury10, 11, 16, 17, and to engraft and re-endothelialize denuded vascular segments or implanted grafts18. In elderly patients and subjects with peripheral arterial disease (PAD) and critical limb ischemia (CLI), circulating or resident ECFCs may become prone to replicative senescence (i.e., ECFCs may lack proliferative potential), thus rendering them impotent for autologous vascular repair. At least for these reasons, it is desirable to find an alternate source of ECFCs that may be used for vascular repair.
Human pluripotent stem cells (human embryonic stem cells and induced pluripotent stem cells, collectively hPSCs) display virtually unlimited self-renewal capacity and ability to differentiate into any cell type in the animal body19-21. Human pluripotent stem cells have been reported to differentiate into cells of the endothelial lineage22-31. However, in vitro hPSC-derived endothelial cells are unstable (e.g., reported to drift to various non-endothelial phenotypes24, 32), exhibit low proliferative potential with a proclivity to reach replicative senescence within 5-7 passages26, 27, 32, and/or lack a capacity for blood vessel formation in vivo in the absence of co-implantation with supportive cells51. There is no published evidence (other than that of the inventors) for in vitro derivation from hPSCs of endothelial cells having proliferative potential equal to or greater than that of cord blood ECFCs (CB-ECFCs) and having the capacity to form blood vessels in vivo in the absence of co-cultured or co-implanted cells.
It is desirable to mitigate and/or obviate one or more of the above deficiencies.